Cytochrome P450 enzymes play a critical role in the metabolism of a wide variety of drugs and other ingested chemical compounds. These enzymes represent the first step in the metabolism of these chemicals; a process that yields products which have very different biological effects from the original compound. A key parameter in the profiling of a candidate drug is the identification of a particular cytochrome P450 enzyme(s) responsible for its metabolism. The objective of this project is to generate simple as well as more sophisticated high throughput screens that will enable individual researchers and large pharmaceutical companies to rapidly identify, in vitro, significant specific P450 drug interactions. Expanding o the success of the Phase I data which demonstrates that human P450s and NADPH reductase can be co-expressed in E coli at high levels, devoid of the intrinsic P450 activities plaguing systems based on eukaryotic expression, Phase II will: (1) generate cytochrome P450 isoforms by their expression in E coli; (2) forma the P450s into simple unambiguous assays that will permit pharmacological and kinetic profiles to be determined in vitro; (3) generate methods based on microcalorimetry and surface plasmon resonance technologies that will determine P450-substrate binding constants; and, (4) adapt the recombinant human P450s into high throughput systems that will form the basis on the Company's drug screening service. PROPOSED COMMERCIAL APPLICATIONS: A comprehensive panel of recombinant, human P450s and human NADPH P450 reductase, in the form of individual reagents, and well as the more "user-friendly" P450-reductase co-expressed membranes will be made available to research and development labs for in vitro drug metabolism studies. These reagents will also be incorporated into an "in-house", custom screening service which will provide high- throughput analysis of P450-drug interactions on a contractual basis.